Limits...
Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure.

Kost N, Kaiser S, Ostwal Y, Riedel D, Stützer A, Nikolov M, Rathke C, Renkawitz-Pohl R, Fischle W - Nucleic Acids Res. (2015)

Bottom Line: This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein.Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation.In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chromatin Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.

Show MeSH

Related in: MedlinePlus

Recombinant Mst77F binds chromatin and induces aggregation. (A) 12 × 200 bp x 601 DNA-based oligonucleosomal arrays were incubated with increasing amounts of Mst77F (none, 0.23–3.71 molar excess over histone octamers). Clustering of protein-chromatin complexes was triggered by addition of MgCl2. Complexes were recovered by centrifugation and analyzed by sodium dodecyl sulphate-PAGE (SDS-PAGE) stained with Coomassie Blue. Input, chromatin and Mst77F (highest concentration) before precipitation. (B) Recombinant 12-mer oligonucleosomal arrays were incubated with the indicated proteins and digested with MNase. DNA was isolated and analyzed on a 1% TBE-agarose gel that was stained with EtBr. Mock, no protein added; input, no MNase added; time, digest for 1, 2, 4 or 8 min. The asterisks mark major (bottom) and minor (top) scavenger DNA species used in the chromatin assembly procedure. (C) AFM images of 12-mer oligonucleosomal arrays in absence (mock) or presence of Mst77F (4-fold molar excess over DNA). Images were recorded in tapping mode; scale bar represents 100 nm. (D) The indicted recombinant proteins were added to nuclei isolated from S2 cells. MNase digest was performed and analyzed as in (B). Mock, no exogenous protein added. (E) MNase digest of 187 bp x 601 DNA-based nucleosome core particles in presence of the indicated recombinant proteins. Mock, no protein added; input, no MNase added; time, digest for 1, 2, 4 or 8 min. DNA was extracted and analyzed on a 5% TBE-PAGE gel that was stained with EtBr.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4381051&req=5

Figure 2: Recombinant Mst77F binds chromatin and induces aggregation. (A) 12 × 200 bp x 601 DNA-based oligonucleosomal arrays were incubated with increasing amounts of Mst77F (none, 0.23–3.71 molar excess over histone octamers). Clustering of protein-chromatin complexes was triggered by addition of MgCl2. Complexes were recovered by centrifugation and analyzed by sodium dodecyl sulphate-PAGE (SDS-PAGE) stained with Coomassie Blue. Input, chromatin and Mst77F (highest concentration) before precipitation. (B) Recombinant 12-mer oligonucleosomal arrays were incubated with the indicated proteins and digested with MNase. DNA was isolated and analyzed on a 1% TBE-agarose gel that was stained with EtBr. Mock, no protein added; input, no MNase added; time, digest for 1, 2, 4 or 8 min. The asterisks mark major (bottom) and minor (top) scavenger DNA species used in the chromatin assembly procedure. (C) AFM images of 12-mer oligonucleosomal arrays in absence (mock) or presence of Mst77F (4-fold molar excess over DNA). Images were recorded in tapping mode; scale bar represents 100 nm. (D) The indicted recombinant proteins were added to nuclei isolated from S2 cells. MNase digest was performed and analyzed as in (B). Mock, no exogenous protein added. (E) MNase digest of 187 bp x 601 DNA-based nucleosome core particles in presence of the indicated recombinant proteins. Mock, no protein added; input, no MNase added; time, digest for 1, 2, 4 or 8 min. DNA was extracted and analyzed on a 5% TBE-PAGE gel that was stained with EtBr.

Mentions: To analyze whether Mst77F has autonomous effects on chromatin or requires cellular cofactors, we set up in vitro systems. First, we analyzed interaction of Mst77F with recombinant oligonucleosomal arrays reconstituted on the 12 × 200 bp x 601 DNA sequence (Figure 2A) (32). In co-precipitation experiments we detected association of the protein with chromatin that could not be saturated at 4-fold molar excess of the protein compared to histone octamers. We also noted that binding of increasing amounts of Mst77F did not cause eviction of any core histone from the chromatin fiber. Next, we analyzed the consequence of Mst77F binding for chromatin conformation. Addition of Mst77F was sufficient to impair MNase digestion of recombinant oligonucleosomal arrays (Figure 2B). While the effect was weaker compared to linker histone hH1.4, imaging of the oligonucleosomal arrays by atomic force microscopy clearly indicated significant chromatin compaction and aggregation induced by Mst77F (Figure 2C).


Multimerization of Drosophila sperm protein Mst77F causes a unique condensed chromatin structure.

Kost N, Kaiser S, Ostwal Y, Riedel D, Stützer A, Nikolov M, Rathke C, Renkawitz-Pohl R, Fischle W - Nucleic Acids Res. (2015)

Recombinant Mst77F binds chromatin and induces aggregation. (A) 12 × 200 bp x 601 DNA-based oligonucleosomal arrays were incubated with increasing amounts of Mst77F (none, 0.23–3.71 molar excess over histone octamers). Clustering of protein-chromatin complexes was triggered by addition of MgCl2. Complexes were recovered by centrifugation and analyzed by sodium dodecyl sulphate-PAGE (SDS-PAGE) stained with Coomassie Blue. Input, chromatin and Mst77F (highest concentration) before precipitation. (B) Recombinant 12-mer oligonucleosomal arrays were incubated with the indicated proteins and digested with MNase. DNA was isolated and analyzed on a 1% TBE-agarose gel that was stained with EtBr. Mock, no protein added; input, no MNase added; time, digest for 1, 2, 4 or 8 min. The asterisks mark major (bottom) and minor (top) scavenger DNA species used in the chromatin assembly procedure. (C) AFM images of 12-mer oligonucleosomal arrays in absence (mock) or presence of Mst77F (4-fold molar excess over DNA). Images were recorded in tapping mode; scale bar represents 100 nm. (D) The indicted recombinant proteins were added to nuclei isolated from S2 cells. MNase digest was performed and analyzed as in (B). Mock, no exogenous protein added. (E) MNase digest of 187 bp x 601 DNA-based nucleosome core particles in presence of the indicated recombinant proteins. Mock, no protein added; input, no MNase added; time, digest for 1, 2, 4 or 8 min. DNA was extracted and analyzed on a 5% TBE-PAGE gel that was stained with EtBr.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4381051&req=5

Figure 2: Recombinant Mst77F binds chromatin and induces aggregation. (A) 12 × 200 bp x 601 DNA-based oligonucleosomal arrays were incubated with increasing amounts of Mst77F (none, 0.23–3.71 molar excess over histone octamers). Clustering of protein-chromatin complexes was triggered by addition of MgCl2. Complexes were recovered by centrifugation and analyzed by sodium dodecyl sulphate-PAGE (SDS-PAGE) stained with Coomassie Blue. Input, chromatin and Mst77F (highest concentration) before precipitation. (B) Recombinant 12-mer oligonucleosomal arrays were incubated with the indicated proteins and digested with MNase. DNA was isolated and analyzed on a 1% TBE-agarose gel that was stained with EtBr. Mock, no protein added; input, no MNase added; time, digest for 1, 2, 4 or 8 min. The asterisks mark major (bottom) and minor (top) scavenger DNA species used in the chromatin assembly procedure. (C) AFM images of 12-mer oligonucleosomal arrays in absence (mock) or presence of Mst77F (4-fold molar excess over DNA). Images were recorded in tapping mode; scale bar represents 100 nm. (D) The indicted recombinant proteins were added to nuclei isolated from S2 cells. MNase digest was performed and analyzed as in (B). Mock, no exogenous protein added. (E) MNase digest of 187 bp x 601 DNA-based nucleosome core particles in presence of the indicated recombinant proteins. Mock, no protein added; input, no MNase added; time, digest for 1, 2, 4 or 8 min. DNA was extracted and analyzed on a 5% TBE-PAGE gel that was stained with EtBr.
Mentions: To analyze whether Mst77F has autonomous effects on chromatin or requires cellular cofactors, we set up in vitro systems. First, we analyzed interaction of Mst77F with recombinant oligonucleosomal arrays reconstituted on the 12 × 200 bp x 601 DNA sequence (Figure 2A) (32). In co-precipitation experiments we detected association of the protein with chromatin that could not be saturated at 4-fold molar excess of the protein compared to histone octamers. We also noted that binding of increasing amounts of Mst77F did not cause eviction of any core histone from the chromatin fiber. Next, we analyzed the consequence of Mst77F binding for chromatin conformation. Addition of Mst77F was sufficient to impair MNase digestion of recombinant oligonucleosomal arrays (Figure 2B). While the effect was weaker compared to linker histone hH1.4, imaging of the oligonucleosomal arrays by atomic force microscopy clearly indicated significant chromatin compaction and aggregation induced by Mst77F (Figure 2C).

Bottom Line: This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein.Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation.In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chromatin Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.

Show MeSH
Related in: MedlinePlus